检测博尔纳病病毒RNA的RACE技术的建立

Establishment of 3′RACE method for detection of Borna disease virus RNA

目的建立检测博尔纳病病毒(BDV)RNA的3′RACE(rapid amplification of cDNA ends)方法。方法根据已知的BDV p40基因序列设计上游引物sp1;提取BDV(H1766株)持续感染OL细胞的总RNA,用引物sp1和oligo dT进行3′RACE扩增,将PCR产物克隆到pGEM-T载体并转化到大肠埃希菌中,制备阳性菌落的目的质粒,进行序列测定和同源性比对;同时对检测BDV RNA的3′RACE方法的特异性和敏感性进行分析。结果建立了检测BDV RNA的3′RACE技术;所获得的BDV p40基因的3′末端扩增产物的核苷酸序列与已知BDV(H1766株)p40基因的核苷酸序列同源性为100%;本方法对BDV RNA(mRNA)具有特异性,但对BDV p40基因重组质粒无扩增结果;并且可以检测到0.04 ng以上含量的BDV感染细胞的总RNA。结论检测BDV RNA的3′RACE技术可以排除实验室污染造成的BDV基因扩增的假阳性,并可用于进一步分析BDV基因序列的特点以及评价BDV相关基因的表达情况。

Objective To establish the 3′RACE method for detection of Borna disease virus （BDV） RNA. Methods Used the BDV p40 gene mRNA specific primer spl and oligo dT, the total RNA of the BDV persistent infecting OL cell were amplified and the positive products were cloned into pGEM-T vector. The positive clones were selected from transformed E. coil,then sequenced and compared with the BDV H1766 strain. Meanwhile the specificity and sensitivity of the method were performed. Results The 3′RACE method for detection of BDV RNA was established and the special fragment of BDV p40 mRNA, with the 100% homogeneity of nucleotide of BDV H1766 strain was obtained; the method manifested specifically to the mRNA of BDV p40 gene but not the BDV p40 gene recombinant plasmid, and might detected the total RNA of BDV infection cells more than 0.04 ng. Conclusion The 3′RACE method for detection of BDV RNA is useful to exclude the false amplification of BDV RNA due to the contamination of plasmids and analyze the characteristic of BDV gene and the transcription of BDV.